The "supercompensation" effect of children's lockdown during COVID-19: based on the analysis of changes in physical activity, sleep, and psychology.

OBJECTIVE: To investigate the "supercompensation" effect of preschoolers during the coronavirus disease 2019 lockdown by comparing the changes in physical activity (PA), psychological, and sleep indicators before and after the lockdown. METHODS: A total of 127 children (aged 3-6 years) were recruited. Before and after the lockdown, the children's PA levels were measured using the ActiGraph GT3X+, and their psychological and sleep indicators were measured using the Strengths and Difficulties Questionnaire (SDQ) and Child Sleep Habit Questionnaire (CSHQ), respectively. RESULTS: Regarding PA, the children's total physical activity, low-intensity physical activity, and medium-intensity physical activity (MVPA) were higher after the lockdown than before the lockdown, with significant differences in MVPA (p < 0.05). Regarding psychology, the children's SDQ and multidimensional scores were better after the lockdown than before the lockdown, with a significant difference in SDQ scores (p < 0.05). Regarding sleep, the children's CSHQ scores were better after the lockdown than before the lockdown, with a highly significant difference in CSHQ scores (p < 0.01). CONCLUSION: After lockdown, children's PA, psychological, and sleep effects were "supercompensated." In particular, the PA of preschoolers before, during, and after the lockdown may show a "baseline-inhibition-supercompensation" process.

Relationship between 24-h activity behavior and body fat percentage in preschool children: based on compositional data and isotemporal substitution analysis.

OBJECTIVE: This study aims to elucidate the dose‒response relationship between 24-h activity behaviors and body fat percentage (BFP) in Chinese preschool children using a compositional isotemporal substitution model (ISM). METHODS: In a cross-sectional design, 881 children aged 3-6 from urban and rural areas of Jiangxi Province were sampled. Activity behaviors, including sedentary behavior (SB), low-intensity physical activity (LPA), and moderate- to high-intensity physical activity (MVPA), were measured using accelerometers. Sleep patterns were assessed through questionnaires, and BFP was determined by bioelectrical impedance analysis (BIA). The study employed compositional data analysis (CoDA) and ISM to estimate the impact of reallocating durations of different activity behaviors on BFP. RESULTS: Higher BFP was found in urban vs. rural children, decreasing with age. Overweight and obesity rates were 10.6% and 7.6%, respectively, above national averages. MVPA and LPA were negatively correlated with BFP, while SB was positively correlated. A 30-min MVPA reduction significantly increased zBFR, particularly in overweight children. Gender-specific nuances revealed that boys' MVPA negatively influenced zBFP (ß = -0.155), P < 0.05), while girls' SB positively impacted zBFP (ß = 0.636, P < 0.01). Isotemporal simulations emphasized amplified effects in overweight children, with boys' zBFR rising rapidly when MVPA was substituted and girls displaying a notable substitution effect between SB and LPA. CONCLUSION: BFP is closely linked to 24-h activity behaviors, notably in overweight and obese preschoolers. ISM identified MVPA as a critical influencer, with a 30-min reduction substantially increasing BFP. Gender disparities were evident, implicating MVPA in boys and LPA and SB in girls.

OsGRP3 Enhances Drought Resistance by Altering Phenylpropanoid Biosynthesis Pathway in Rice (Oryza sativa L.).

As a sessile organism, rice often faces various kinds of abiotic stresses, such as drought stress. Drought stress seriously harms plant growth and damages crop yield every year. Therefore, it is urgent to elucidate the mechanisms of drought resistance in rice. In this study, we identified a glycine-rich RNA-binding protein, OsGRP3, in rice. Evolutionary analysis showed that it was closely related to OsGR-RBP4, which was involved in various abiotic stresses. The expression of OsGRP3 was shown to be induced by several abiotic stress treatments and phytohormone treatments. Then, the drought tolerance tests of transgenic plants confirmed that OsGRP3 enhanced drought resistance in rice. Meanwhile, the yeast two-hybrid assay, bimolecular luminescence complementation assay and bimolecular fluorescence complementation assay demonstrated that OsGRP3 bound with itself may affect the RNA chaperone function. Subsequently, the RNA-seq analysis, physiological experiments and histochemical staining showed that OsGRP3 influenced the phenylpropanoid biosynthesis pathway and further modulated lignin accumulation. Herein, our findings suggested that OsGRP3 enhanced drought resistance in rice by altering the phenylpropanoid biosynthesis pathway and further increasing lignin accumulation.

Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance.

PURPOSE: Tryptophan's (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. METHODS: Selective Trp oxidation was induced by 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. RESULTS: Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. CONCLUSIONS: Trp oxidation in CDRs can adversely impact mAbs' conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.

Ultrasmall Bi/Cu Coordination Polymer Combined with Glucose Oxidase for Tumor Enhanced Chemodynamic Therapy by Starvation and Photothermal Treatment.

Multi-modal combination therapy for tumor is expected to have superior therapeutic effect compared with monotherapy. In this study, a super-small bismuth/copper-gallic acid coordination polymer nanoparticle (BCN) protected by polyvinylpyrrolidone is designed, which is co-encapsulated with glucose oxidase (GOX) by phospholipid to obtain nanoprobe BCGN@L. It shows that BCN has an average size of 1.8 ± 0.7 nm, and photothermal conversion of BCGN@L is 31.35% for photothermal imaging and photothermal therapy (PTT). During the treatment process of 4T1 tumor-bearing nude mice, GOX catalyzes glucose in the tumor to generate gluconic acid and hydrogen peroxide (H2 O2 ), which reacts with copper ions (Cu2+ ) to produce toxic hydroxyl radicals (•OH) for chemodynamic therapy (CDT) and new fresh oxygen (O2 ) to supply to GOX for further catalysis, preventing tumor hypoxia. These reactions increase glucose depletion for starvation therapy , decrease heat shock protein expression, and enhance tumor sensitivity to low-temperature PTT. The in vitro and in vivo results demonstrate that the combination of CDT with other treatments produces excellent tumor growth inhibition. Blood biochemistry and histology analysis suggests that the nanoprobe has negligible toxicity. All the positive results reveal that the nanoprobe can be a promising approach for incorporation into multi-modal anticancer therapy.

Effects of structured and unstructured interventions on fundamental motor skills in preschool children: a meta-analysis.

Background: It has been suggested that higher levels of fundamental motor skills (FMS) promote the physical health of preschool-aged children. The impacts of structured and unstructured interventions on FMS in children aged 10-16 years have been widely acknowledged in previous studies. However, there is a lack of relevant studies in preschool-aged children. Objective: This meta-analysis aimed to compare the effects of structured and unstructured interventions on FMS in preschool-aged children. Methods: The PubMed, Web of Science, and Google Scholar databases were searched from inception to 1 November 2023 to identify experiments describing structured and unstructured interventions for FMS in preschool-aged children. The Downs and Black Checklist was used to assess the risk of bias. A random effects model was used for the meta-analysis to evaluate the pooled effects of interventions on FMS. Subgroup analyses based on the duration and characteristics of the intervention were conducted to identify sources of heterogeneity. Results: A total of 23 studies with 4,068 participants were included. There were 12 studies examining structured interventions, 9 studies examining unstructured interventions, and 6 studies comparing structured vs. unstructured interventions. The risk of bias in the included studies was generally low. All interventions significantly improved FMS in preschool-aged children compared to control treatments (p < 0.05). Structured interventions had more significant effects on locomotor skills (LMSs) in preschool-aged children than unstructured interventions (Hedges' g = 0.44, p = 0.04). The effects of structured interventions were strongly influenced by the total intervention duration, such that long-term interventions were more effective (Hedge's g = 1.29, p < 0.001). Conclusion: Structured interventions play a crucial role in enhancing FMS among young children, especially when considering LMSs. These interventions require consistent and repeated practice over time to reach proficiency. Systematic review registration: PROSPERO, identifier number CRD42023475088, https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023475088.

Structured-unstructured activity behaviours on preschoolers' mental and physical health: a compositional and isotemporal reallocation analysis.

This study analysed the relationship between the structured and unstructured activities of preschoolers and their mental and physical health, and also investigated the predicted changes in mental and physical health by reallocating activity time. This cross-sectional study was carried out with 324 preschoolers. Video recording and SOPARC activity observation system was used for the division of structured and unstructured activities. An accelerometer sensor was used to measure activity intensity. The SDQ psychological questionnaire was adopted to collect data on internalizing difficulties, externalizing difficulties, total difficulties and pro-social behaviours. Physical indices including body shape (height, weight, BMI), and physical fitness (upper and lower limb strength, flexibility, agility, and balance) were collected using Chinese toddler physical fitness measurement tools. Component data and isotemporal reallocation analyses were conducted using R Studio (Version 4.2). A total of 308 preschoolers (160 boys; aged 4.50 ± 0.93 years) were included in the data analysis. The activities composition, adjusted for sex, area, mental level (for mental indicators), or age (for physical indicators), was significantly correlated with various measurement indicators (p < 0.05). Specifically, structured (ß=-0.87, p < 0.05) and unstructured (ß=-1.24, p < 0.05) moderate-to-vigorous physical activity (MVPA) were significantly positive correlated with internalizing difficulties, while structured MVPA was significantly positively correlated with body shape (ß = 2.17, p < 0.05). Replacing structured light physical activity (LPA) with 10 min of structured MVPA has a positive effect on internalizing difficulties (SMD=-1.28, 95%CI: -2.30 to -0.27) and body shape (SMD = 1.76, 95%CI: 0.37 to 3.15). When the total replacement time reaches 25 min, the benefits become even more pronounced. Structured and unstructured MVPA are both beneficial to preschoolers' mental and physical health, with the incorporation of MVPA for over 25 min in structured activities and supplementary unstructured MVPA yielding even greater benefits.

A cohort study of factors influencing the physical fitness of preschool children: a decision tree analysis.

Objective: Based on the decision tree model, to explore the key influencing factors of children's physical fitness, rank the key influencing factors, and explain the complex interaction between the influencing factors. Methods: A cohort study design was adopted. 1,276 children (ages 3-6) from 23 kindergartens in Nanchang, China, were chosen for the study to measure the children's physical fitness at baseline and a year later and to compare the physical fitness scores at the two stages. The study was conducted following the Chinese National Physical Fitness Testing Standard (Children Part); To identify the primary influencing factors of changes in physical fitness, a decision tree model was developed, and a questionnaire survey on birth information, feeding patterns, SB, PA, dietary nutrition, sleep, parental factors, and other relevant information was conducted. Results: The levels of physical fitness indicators among preschool children showed a significant increase after 1 year. The accuracy of the CHAID model is 84.17%. It showed that 7 variables were strongly correlated with the physical changes of children's fitness, the order of importance of each variable was weekend PA, weekend MVPA, mother's BMI, mother's sports frequency, father's education, mother's education, and school day PA. Three factors are related to PA. Four factors are related to parental circumstances. In addition to the seven important variables mentioned, variables such as breakfast frequency on school day, puffed food, frequency of outing, school day MVPA, parental feeling of sports, father's occupation, and weekend breakfast frequency are all statistically significant leaf node variables. Conclusion: PA, especially weekend PA, is the most critical factor in children's physical fitness improvement and the weekend MVPA should be increased to more than 30 min/d based on the improvement of weekend PA. In addition, parental factors and school day PA are also important in making decisions about changes in fitness for children. The mother's efforts to maintain a healthy BMI and engage in regular physical activity are crucial for enhancing the physical fitness of children. Additionally, other parental factors, such as the parents' educational levels and the father's occupation, can indirectly impact the level of physical fitness in children.

Genome-wide identification and comprehensive analysis of tubby-like protein gene family in multiple crops.

Introduction: The highly conserved tubby-like proteins (TLPs) play key roles in animal neuronal development and plant growth. The abiotic stress tolerance function of TLPs has been widely explored in plants, however, little is known about comparative studies of TLPs within crops. Methods: Bioinformatic identification, phylogenetic analysis, Cis-element analysis, expression analysis, Cis-element analysis, expression analysis and so on were explored to analysis the TLP gene family of multiple crops. Results: In this study, a comprehensive analysis of TLP genes were carried out in seven crops to explore whether similar function of TLPs in rice could be achieved in other crops. We identified 20, 9, 14, 11, 12, 35, 14 and 13 TLP genes in Glycine max, Hordeum vulgare, Sorghum bicolor, Arabidopsis thaliana, Oryza sativa Japonica, Triticum aestivum, Setaria italic and Zea mays, respectively. All of them were divided into two groups and ten orthogroups (Ors) based on amino acids. A majority of TLP genes had two domains, tubby-like domain and F-box domain, while members of Or5 only had tubby-like domain. In addition, Or5 had more exons and shorter DNA sequences, showing that characteristics of different Ors reflected the differentiated function and feature of TLP genes in evolutionary process, and Or5 was the most different from the other Ors. Besides, we recognized 25 cis-elements in the promoter of TLP genes and explored multiple new regulation pathway of TLPs including light and hormone response. The bioinformatic and transcriptomic analysis implied the stresses induced expression and possible functional redundancy of TLP genes. We detected the expression level of 6 OsTLP genes at 1 to 6 days after seed germination in rice, and the most obvious changes in these days were appeared in OsTLP10 and OsTLP12. Discussion: Combined yeast two-hybrid system and pull down assay, we suggested that the TLP genes of Or1 may have similar function during seed germination in different species. In general, the results of comprehensive analysis of TLP gene family in multiple species provide valuable evolutionary and functional information of TLP gene family which are useful for further application and study of TLP genes.

Effects of Family Intervention on Physical Activity and Sedentary Behavior in Children Aged 2.5-12 Years: A Meta-Analysis.

Introduction: To use a quantitative approach to examine the effects of family interventions on physical activity (PA) and sedentary behavior (SB) in children aged 2. 5-12 years. Methods: PubMed, OVID, Web of Science, and others were searched from their inception to May 2020. Intervention studies that examined the effects of family interventions on PA among children aged 2.5-12 years were included in this meta-analysis. Lastly, subgroup analyses were conducted to examine the potential modifying effects of family intervention's characteristics and study quality. Results: Eleven articles met the inclusion criteria for this review. Studies investigated a range of PA outcomes, including moderate-to-vigorous PA (MVPA), total PA (TPA), daily steps, and SB levels. Meta-analysis showed that family intervention had a significant effect on PA [standardized mean difference (SMD) = 0.10; 95% CI = 0.01-0.19], especially for daily steps [weight means difference (WMD) = 1,006; 95% CI = 209-1,803], but not for SB (WMD = -0.38; 95% CI = -7.21-6.46). Subgroup analyses indicated the improvements in PA occurred when children were 6-12 years old, intervention focused on PA only, intervention duration ≤ 10 weeks, and "low risk of bias" study performed. Conclusions: Family intervention may be a promising way to promote children's PA levels, especially for daily steps. Trial Registration: Meta-analysis protocol was registered on PROSPERO: CRD42020193667.

Effects of Smartphone-Based Interventions on Physical Activity in Children and Adolescents: Systematic Review and Meta-analysis.

BACKGROUND: About 70% of children and adolescents worldwide do not meet the recommended level of physical activity (PA), which is closely associated with physical, psychological, and cognitive well-being. Nowadays, the use of technologies to change PA is of interest due to the need for novel, more effective intervention approaches. The previous meta-analyses have examined smartphone-based interventions and their impact on PA in adults, but evidence in children and adolescents still needs further research. OBJECTIVE: This systematic review and meta-analysis aimed to determine the effectiveness of smartphone-based interventions for improving PA in children and adolescents. METHODS: Five electronic databases (PubMed, Web of Science, OVID, Scopus, and the China National Knowledge Infrastructure) were searched up to June 29, 2020. Randomized controlled trials with a control group that examine the effect of smartphone interventions on PA among children and adolescents were included. Bias risks were assessed using the Cochrane collaboration tool. Meta-analysis was performed to assess the pooled effect on PA using a random effects model. Subgroup analyses were conducted to examine the potential modifying effects of different factors (eg, types of intervention, intervention duration, age, measurement, study quality). RESULTS: A total of 9 studies were included in this review, including 4 mobile app interventions, 3 SMS text messaging interventions, and 2 app + SMS text messaging interventions. In general, the risk of bias of included studies was low. Compared with the control group, the use of smartphone intervention significantly improved PA (standardized mean difference [SMD] 0.44, 95% CI 0.11-0.77, P=.009), especially for total PA (TPA; weighted mean difference [WMD] 32.35, 95% CI 10.36-54.33, P=.004) and daily steps (WMD 1185, 95% CI 303-2068, P=.008), but not for moderate-to-vigorous PA (WMD 3.91, 95% CI -1.99 to 9.81, P=.19). High statistical heterogeneity was detected (I2=73.9%, P<.001) for PA. Meta-regression showed that duration (ß=-.08, 95% CI -0.15 to -0.01, n=16) was a potential factor for high heterogeneity. The results of subgroup analyses indicated that app intervention (SMD 0.76, 95% CI 0.23-1.30, P=.005), children (SMD 0.64, 95% CI 0.10-1.18, P=.02), "≤8 weeks" (SMD 0.76, 95% CI 0.23-1.30, P=.005), objective measurement (SMD 0.50, 95% CI 0.09-0.91, P=.02), and low risk of bias (SMD 0.96, 95% CI 0.38-1.54, P=.001) can significantly improve PA. CONCLUSIONS: The evidence of meta-analysis shows that smartphone-based intervention may be a promising strategy to increase TPA and steps in children and adolescents. Currently, app intervention may be a more effective strategy among smartphone intervention technologies. To extend the promise of smartphone intervention, the future needs to design comparative trials among different smartphone technologies. TRIAL REGISTRATION: PROSPERO CRD42019148261; https://tinyurl.com/y5modsrd.

Changes in Chinese Adults' Physical Activity Behavior and Determinants before and during the COVID-19 Pandemic.

PURPOSE: To investigate the changes in Chinese adults' physical activity (PA) behavior and determinants before and during the COVID-19 pandemic. METHOD: A total of 1028 adults (aged 19-59 years) were recruited from 127 urban and rural neighborhoods in China using stratified three-stage probability sampling. Data collection was conducted in December 2019 and July 2020. RESULTS: Compared with the data before the pandemic, individuals' weekly moderate-to-vigorous-intensity PA (MVPA) decreased significantly from 139 min to 120 min, seven months after the outbreak (p = 0.01), with female and rural populations displaying a more significant decrease (p = 0.02). Overall, 13.7% of participants met the PA guidelines (World Health Organization) both before and during the pandemic, while 21.8% met the guidelines only before the pandemic and 18.1% increased their PA and met the PA guidelines during the pandemic. A total of 46.4% did not meet the PA guidelines before or during the pandemic. Determinants of PA behavior change before and during the pandemic included sports skills, self-determined motivation and support from sports organizations. CONCLUSIONS: The Chinese adults' PA levels decreased significantly from before to during the COVID-19 pandemic, particularly among the female population. It is suggested that the enhancement of self-determined motivation, improvement of sport skills, and support from sports organizations might be effective in facilitating individuals' engagement in PA during the pandemic.

Dramatically Enhanced Immunochromatographic Assay Using Cascade Signal Amplification for Ultrasensitive Detection of Escherichia coli O157:H7 in Milk.

The conventional colloidal gold immunochromatographic assay (AuNP-ICA) cannot meet the requirements for the rapid and sensitive detection of Escherichia coli (E. coli) O157:H7 because of its poor sensitivity. Herein, a novel two-step cascade signal amplification strategy that integrates in situ gold growth and nanozyme-mediated catalytic deposition was proposed to enhance the detection sensitivity of conventional AuNP-ICA dramatically. The enhanced strip displayed ultrahigh sensitivity in E. coli O157:H7 detection and had a detection limit of 1.25 × 101 CFU/mL, which is approximately 400-fold lower than that of traditional AuNP-ICA (5 × 103 CFU/mL). The amplified strip had no background signal in the T-line zone in the absence of E. coli O157:H7 even after one round of cascade signal amplification. The enhanced strip demonstrated excellent selectivity against E. coli O157:H7 with a negligible cross-reaction to nine other common pathogens. Intra-assays and interassays showed that the improved strip has acceptable accuracy and precision for determining E. coli O157:H7. The average recoveries in a real milk sample ranged from 87.33 to 112.15%, and the coefficients of variation were less than 10%. The enhanced strip also showed great potential in detecting a single E. coli O157:H7 cell in a 75 µL solution.

Effects of HIIT and MICT on cardiovascular risk factors in adults with overweight and/or obesity: A meta-analysis.

OBJECTIVE: The purpose of this study was to evaluate the effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on cardiovascular disease (CVD) risk factors in adults with overweight and obesity. METHODS: Twenty-two articles were included by searching six databases, the total number of subjects was 620 in these articles. Outcomes were synthesised using a random-effects meta-analysis of the Standardized mean difference (SMD) in CVD risk factors. RESULTS: HIIT and MICT resulted in statistically significant reductions in Weight, BMI, fat%, total cholesterol(TC), and improvement in VO2max. Compared with MICT, subgroup of durations of HIIT training interval ≥2 min can significantly increase VO2max (SMD = 0.444, 95% CI:0.037~0.851,P = 0.032), subgroup of energy expenditure of HIIT equal to MICT can significantly increase VO2max (SMD = 0.399, 95% CI:0.106~0.692,P = 0.008). CONCLUSIONS: HIIT appears to provide similar benefits to MICT for improving body composition, VO2maxand TC, but HIIT spent less time than MICT by 9.7 min on one session. HIIT is superior to MICT in improving cardiopulmonary fitness when durations of HIIT training interval ≥2 min or energy expenditure of HIIT same as MICT. PROSPERO ID: CRD42016045835.

Gold Nanoflower-Enhanced Dynamic Light Scattering Immunosensor for the Ultrasensitive No-Wash Detection of Escherichia coli O157:H7 in Milk.

Gold nanoflowers (GNFs) exhibit stronger light scattering ability than gold nanospheres (GNSs) with the same diameter, thereby contributing to enhancing the sensitivity of the scattering-based sensing method. However, the application of GNFs in biosensors based on dynamic light scattering (DLS) has not been yet reported. Herein, we describe for the first time an improved no-wash immunosensor based on dynamic light scattering for the detection of Escherichia coli O157:H7 (E. coli O157:H7) in milk using GNFs for sensitive signal transduction. To achieve this goal, a thiolated amphiphilic carboxyl ligand was introduced to modify the GNF surface and improve solution stability and antibody functionalization. Several key factors that affect the detection sensitivity of our developed GNF_DLS immunosensor were systematically investigated. Under the optimal conditions, our proposed GNF_DLS immunosensor provided an excellent linear detection for E. coli O157:H7 within the range from 6 × 100 to 6 × 104 colony-forming units (CFU)/mL, with a limit of detection of 2.7 CFU/mL. Combined with our previously reported two-step large-volume immunomagnetic separation (IMS) method, the designed GNF_DLS immunosensor can sensitively, selectively, and accurately detect the presence of E. coli O157:H7 in pasteurized milk. The potential of our GNF_DLS method for monitoring the presence of a single bacterial cell in 1 mL of sample solution was also demonstrated. Overall, the developed GNF_DLS immunosensor can be used for the rapid and high-sensitivity determination of pathogenic bacteria and can be extended for the ultrasensitive no-wash detection of other trace analytes.

[Effects of different intensity exercise training on apoptosis-related microRNAs and the targeted proteins in cardiomyocytes].

OBJECTIVE: To detect the levels of miR-1, miR-21 and their targeted proteins in hearts of mice after different exercise training, and discuss potential molecular mechanism. METHODS: Male C57BL/6 mice were randomly divided to 3 groups:sedentary (SE), exercise training 1(ET1) and exercise training 2 (ET2). SE did not do any exercise; ET1 undertook swimming training for 8 weeks, once a day, 5 days/week. Swimming 30 min in the 1st week, and the duration was increased 10 min per week to 90 min and maintained in the 7th and 8th week. ET2 performed the same work as ET1 and switched to twice a day by the end of the 5th week. TUNEL assay was applied to test myocardial apoptosis. Western blot and RT-PCR were used to detect proteins and miRs levels respectively. RESULTS: Compared with SE, in ET1, myocardial apoptosis and miR-1 level did not change, but its targeted protein Bcl-2 increased significantly(P<0.01). miR-21 and its targeted protein PDCD4 did not change significantly. In ET2, myocardial apoptosis and miR-1 level were decreased significantly(P<0.05). Bcl-2 was increased significantly(P<0.01). miR-21 also increased significantly (P<0.05), but PDCD4 did not decrease significantly. CONCLUSIONS: Exercise training in ET2 other than ET1 could down-regulate myocardial apoptosis. Alterations of miR-1 and Bcl-2 may be responsible for this cardioprotection. PDCD4 is not sensitive to exercise training, it is likely that miR-21 and other targeted proteins participate in exercise-regulative apoptosis.

Identification and quantification of signal peptide variants in an IgG1 monoclonal antibody produced in mammalian cell lines.

Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.

Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells.

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.

Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines.

Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping.

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (∼0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.